PROJECT SUMMARY Glioblastoma (GBM) is an intractable cancer with an average survival time of 12 to 15 months. Treatment options are limited by the infiltration of tumor cells into healthy tissue and the difficulty of delivering effective chemotherapeutics across the blood brain barrier, but engineered neural stem cells (NSCs) hold great promise as GBM therapies because they selectively migrate to tumor cells and can be modified to deliver chemotoxic agents directly to those cells. The concept has been demonstrated in multiple studies, and several clinical trials are underway with engineered human allogenic NSCs. However, the use of allogenic cells requires immunosuppression and may reduce the persistence and efficacy of the treatment. Engineered autologous NSCs could overcome these challenges and further improve the efficacy of this technology. With the support of a previous Phase I STTR award, Falcon Therapeutics Inc. and Dr. Shawn Hingtgen at the University of North Carolina?Chapel Hill used a novel transdifferentiation (TD) strategy to develop the first induced NSC-based drug carriers derived from the skin of patients with GBM (iNSCTE). Falcon demonstrated that 1) its single-factor SOX2 TD strategy converted human skin fibroblasts into tumor-homing early-stage induced NSCs (h-iNSCTE); 2) h-iNSCTE rapidly migrated to human GBM cells and penetrated human GBM spheroids without inducing stem-cell based tumors; 3) h-iNSCTE thymidine kinase/ganciclovir enzyme/prodrug therapy (h-iNSCTE?TK) reduced the size of patient-derived GBM xenografts by 95% and extended survival from 32 to 62 days; and 4) h-iNSCTE?TK therapy delivered into the postoperative GBM surgical resection cavity delayed the regrowth of residual GBMs and prolonged survival from 28 to 62 days. These results demonstrated that TD of human skin into h-iNSCTE is a viable platform for creating tumor-homing cytotoxic cell therapies for cancer, where the potential to avoid carrier rejection could maximize treatment durability in human trials. The studies proposed in Phase II will advance h-iNSCTE?TK therapy to the pre-IND stage: Aim 1) Develop a cGMP-compatible process for h-iNSCTE?TK production; Aim 2) Conduct an IND-enabling toxicity, biodistribution, and tumorigenicity study in immunodeficient NSG mice; and Aim 3) Determine the efficacy of intracerebroventricular re-dosing of h- iNSCTE?TK in human GBM xenografts. Phase II metrics of success are to 1) develop a safety package that will be submitted to the FDA as part of an IND filing and 2) confirm the efficacy h-iNSCTE?TK therapy re-dosing in a clinically relevant setting. The Phase II mechanism is appropriate based on a) successful completion of Phase I milestones and demonstration of the mechanism of action and in vivo safety and efficacy, b) the fact that multiple Phase I clinical tests have shown the safety of allogeneic NSCs, and c) the dire need for an FDA- approved product to improve survival outcomes for GBM. The study will support further clinical development of this innovative, high impact approach to autologous iNSC therapy.